366 Honn. Metab. Res. 11 (1979) 366 370
Propranolol Inhibits the In Vitro Conversion of Thyroxine into Triiodothyronine by Isolated Rat Liver Parenchymal Cells C.J. F. van Noorden, W.M. Wiersinga and J. L. Touber Division of Endocrinology, Department of Medicine, University Hospital "Wilhelmina Gasthuis", Amster· dam, The Netherlands
vel of T3 in hyperthyroid patients and in hypothyroid patients on L-thyroxine substitution therapy (WierA model for the in vitro study of the conversion of thyroxsinga and Touber 1977). Again, inhibition of the peine into trüodothyronine using isolated rat liver parenchymal ripheral conversion seemed the most likely explanacells is described. Isolated liver cells (mean pro tein content 18 mg/mI) convert tion for tbis finding. Since the liver is a major site approximately 0.8% of 1.3 /.IM exogenously added T4 into of T4 -deiodination the decrease in plasma TJ - and T3 during thirty minutes incubation. Carbimazole (50 /.IM) presumably, in T3 production - could be caused by has no effect on the conversion process, whereas propylthia direct effect of propranolol on the parenchym al ouracil (50 /.IM) inhibits the conversion, The tI-adrenoceptor blocking agent propranolol lowers the conversion ratio when liver cells. However, a decrease in T3 production as added in concentrations of 580 and 1160 /.IM, but has no in- a consequence of a decrease in liver perfusion caushibitory effect when 290 j.!M is added. ed by propranolol (Nies and Shand 1975) seemed equally possible. In order to further elucidate the Key-Words: Jsolated Rat Liver Cells - Propranolol - Con· action of propranolol, we studied the effect of the version of Thyroxine - Triiodothyronine drug on liver cells in vitro. The isolated liver cell Introduction preparation - rather than liver cell homogenate was chosen because the action of propranolol is melt has been demonstrated conclusively that 3,3',5-triiodothyronine (T3 ) is mainly produced extrathyroid- dia ted via blockade of the ß-adrenergic receptors of the liver cell plasma membrane (Wolfe, Harden and ally by deiodination of thyroxine (T4 ). This process, Molino!f 1976). known as the peripheral conversion of T4 , leads to formation of the metabolically active hormone TJ and to the inactive 3,3',5' -T3 (reverse T3 or rTJ ) flJra- Materials and Methods verman, Ingbar and Sterling 1970; Schwartz, Surks Liver cells of two groups of male Wistar rats were used; and Oppenheimer 1971; Fisher, Chopra and Dussault mean weight ± SD are 215 :t 11 g in group A (n = 4) and 1972; Chopra 1976; Westgren, Melander, Ingemans298 :t 18 g in group B (n = 12). The rats were anaesthetized with nembutal and received intravenously 0.2 ml heparin son, Burger, Tibblin and W/lhlin 1977). In man seve(1000 i.u.) dissolved in 0.8 ml 1% NaN02 to prevent blood ral dru~s have been shown to influence the periphecoagulation and vascular contraction. Parenchymalliver cells ral conversion. Propyltbiouracil (Abuid and Larsen were isolated by perfusion of the liver with calcium-free 1974; Saberi, Sterling and Utiger 1975; Geffner, Azu- phosphate buffered Ringer-solution saturated with carbogen kizawa and Hersh17um 1975; Westgren, Melander, Wa- (pH 7.4, 37°C) containing 20 g/I bovine serum albumin lin and Lindgren 1977), dexamethasone (Chopra, Wil- and 0.6. g/I collagenase (Sigma) (James 1977). The isolated cells were suspended in 3 ml of a Krebs-Ringer-bicarbonate liams, Orgiazzi and Solomon 1975; Bu", Ramsden, buffer (pH 7.4, 37°C, containing 2.5 g/I bovine serum alGriffiths, Black, Hoffenberg, Meinhold and Wenzel bumin but no collagenase) at a concentration of 10-20 mg 1976; Westgren, Ahren, Burger, Ingemansson and protein per ml and incubated at 37 oe in slowly rotating tubes. Cell suspensions of each individual rat of group A Melander 1977), amiodarone (Burger, Dinichert, Niwere incubated in duplicate with 1.3 /.IM sodium-L-thyroxcod, Jenny, Lemarchand-Beraud and Valloton 1976), ine (Sigma) in the presence or absence of 50 /.IM carbimaand Na-iopanoate (Bürgi, Wimpjheimer, Burger, zole, 50 /.IM 6-propy1-2-thiouracil or 580 /.IM D,L-propranZaunbauer, Rösler and Lemarchand-Beraud 1976), 0101 (I.c.I.). In the same manner, cells of the rats of group all decrease plasma levels of TJ with a concomitant B were incubated with 1.3 /.IM sodium L-thyroxine with or rise in plasma rTJ . Also, propylthiouracil inhibits without D,L-propranolol at concentrations of 290, 580 and 1160 /.IM respectively. the conversion ofT4 into T3 in rat liver homogenate in vitro (Visser, van der Does-Tobe, Docter and HenT4 and the various drugs were added together to the incubaUon mixture and 500 /.11 sampies were taken from the inneman 1975; Chopra 1977). We have recently demonstrated that the ß-adrenoceptor blocking agent propranolol lowers the plasma leReceived: 21 June 1978 0018-5043/79
0532-0366
cubation mixtures at times 0, 15, 30 and 60 minute!. The sampies were immediately transfened to I ml ice-cold absolute ethanol After centrifugation (2000 g, 15 min, 4°q the clear supernatants were stored at -20°C. As controls,
Accepted: 29 Aug. 1978 S 03.00
©
1979
Georg Thieme Publishers
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Summuy
Inhibition of in Vitro Conversion of T4 to T3 by Propanolol in Isolated Rat Liver CeUs
367
Table 1 Effect of carbimazole, propylthiouracil and propranolol on the conversion of exogenous T4 (1.3 J'M) into T3 by iso 1ated rat liver cells. Conversion ratios are given as the mean ± SEM (group A, n = 4; mean pro tein content ± SD is 18.9±3.8 mg/mI)
15 30 60
controls
conversion ratio (nm»1 T3 formed / nmol T4 added x 100 %) carbimazole PTU propranolol 50 11M 50 11M 580 11M
0.49 ± 0.10 0.86 ± 0.20 1.06 ± 0.25
-0.05 ± 0.02 -0.03 ± 0.06 -0.03 ± 0.06
0.39 ± 0.13 0.78 t 0.18 1.10 t 0.06
100 ,
liver cells were incubated in the absence of T4, alrld T4 was incubated in the absence of liver cells. The protein concentration of the incubation mixture was determinecl by the biuret method (Itzhaki and Gi111964); the concenlration served as a measure of the number of cells present.
90
!
The T3 and T4 concentrations of the ethanol extracts (diluted if necessary) were measured in duplicate by spedfic radio- Bo immunoassays Wisser et al. 1975; Chopra 1977), using 300 g/l polyethyleneglycol (Cheung and Haunwhite lL 1976) for the separation of the bound and free hormone fuctions. Sampies presenting the standard curve were incubated under identica1 conditions, i.e. with a fmal ethanol conl:entration of 6.7% (v/v). The detection limit of the T3-assar is 0.05 nM, of the T4-assay 1.5 nM. Cross-reactivity of T4 in the T3-assay is 0.1). However, both liver cells; in general approximately 10 nM T3 is 50 ILM propylthiouracil and 580J-LM propranolollowered conversion ratios (p < 0.01). formed after 30 min. incubation.
368
C.J.F. van Noorden, W.M. Wiersinga and J.L. Touber
Table 2 Effect of various concentrations of propranolol on the conversion of exogenous T4 (1.3 "M) into T3 by isolated . rat liver cells. Conversion ratiosaregivenas themean ± SEM (Jroup B, n = 12; mean protein content ± SD is 18.1 ± 1.1 mg/mI) Incubation time in minutes
15 30 60
conversion ratio (nmol T3 formed / nmol T4 added x 100%) propranolol propranolol propranolol 290 pM 580 "M 1160 "M
controls 0.52 0.82 1.47
± ± ±
0.06 0.11 0.20
0.34 0.69 1.46
± ± ±
0.21 ± 0.02 0.55 ± 0.10 1.11 :t 0.13
0.05 0.12 0.16
•
1.5
l
0.26:t 0.05 0.56:t 0.09 0.99:t 0.16
1.5
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0.5
o
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30
80
90 TIME (minut•• )
120
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30
TIME (minut•• )
Fig. 2 Conversion of exogenous T4 (1.3 "M) into T, by OOlated rat liver cells (pro tein content 7.9 mg/ml)
Fig. 3 Conversion of exogenous T4 (l.3·"M) into T3 by 001ated rat liver cells (group A, n =4; mean protein content ± SD is 18.9 ± 3.8 mg/mi). Conversion ratios are given as mean :t SEM.• - . controls, . - . carbimazole 50"M, 0 - 0 proprylthiouracil SO"M, . - . propranolol S80 "M.
No interactions are found in the experiments of group B (results are given in Table 2 and Figure 4). The interactions - and residual sum of squares were pooled. The analysis was continued by a stepwise multiple comparison procedure for ordered parameters (Spj,tvoll 1977). The level of significance was taken as a=0.05. Addition of 580 and 1160 llM propranolol both resulted in lower conversion ratios, but no difference was found between the 580 and 1160 llM dose. No inhibitory effect on the conversion was found for 290 llM propranolol.
when T4 is incubated without cells. We have applied conversion ratios because the deiodinatins capacity of the liver cells is not saturated at the T4 concentrations used (Hesch, Brunner and Söling 1975;
Discussion Our results indicate that isolated rat liver cells are suitable for the in vitro study of the conversion of T4 into T3 ; T3 generation cannot be detected in the absence of T4 nor is there a change in T3 level
Chopra 1977). Conversion of T4 into T3 by isolated liver cells has been reported previously by Hesch, Brunner and Söling (1975). Using a comparable amount of cells, they found a lower conversion ratio (0.1 %) than in the present study (0.8%). Rat liver homogenate has also been shown to generate T3 from T4 (Visser et a1. 1975; Hüfner and Knöpfte 1976; Visser, van der Does-Tobe, Docter and Hennmuzn 1976; Chopra 1977; Hüfner, Grussendorf and Ntokalou 1977); conversion ratios in this model were somewhat higher dependent on the concentrations of substrate and (cell)protein.
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u
Inhibition of in Vitro Conversion of T4 to T3 by Propranolol in Isolated Rat Liver Cells
The results of the experiments with the eells of the rats of JrQuP A show quite c1early that whc,reas the addition of earbimazole has no effeet on the eonversion process, propylthiouraeil inhibits Tl-generation. Ifl. This is in aeeordanee with the results of studies using ;; rat Iiver homogenate (Visser et a1. 1975; Chopra 1977)_ ~
369
1.5
a:
An effeet of propranolol on the in vitro eonversion of T4 has thus far not been reported. Our data show that tbis ß-adrenoceptor blocking agent also inhibits the eonversion of T4 into Tl by isolated rat liver cells. The effeet of propranolol is not an artefact, sinee the drug did not influenee either the T3 levels of eells ineubated without T4 or the radioimmunoassays of T3 and T4 • We have been unable to establish an unequivocal dose dependent effeet of proprano101. Alth()ugh both 580 and 1160 11M propranolol inhibit the eonversion process while 290 11M propranolol does not, there is no difference between the effeets of 580 alld 1160
z
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I
o
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0.5
2
Sinee propranolo1 is rapidly metabolised by the 1iver (Nies and Shand 1975) low levels of Jlroprano101 may not be suffieient to initiate or to maintain an inhibitory effect. Tbis possibility might be explored in experiments using ß-adrenoceptor blocking agents wbieh are not metabolised by the liver, such as atenolol and praetolol. The eoncentrations of propranolol used in our ineubation mixtures may have been mueh bigher than in vive portal plasma levels of subjeets given the usual therapeutie dosage. The results of the studies of Hayes and Cooper (1971) indicate that, in the dog, propranolol eoneentrations in liver tis~rue are in the order of 0.5 Ilg/g (about ten times bigher than the eorresponding blood levels) after the administration of 1 mg/kg orally. It is therefore eonceivab1e that in our experiments other than the ß-blocking properties of the drug - such as the membrane stabilising aetivity - may have influeneed the results. Studies with ß-b1ocking agents whieh lack tbis aetivity (e.g. atenolol and metoprolol) are required to further elueidate the meehanism of action in this respect_ In the rats of group A proprano101 seems t() inhibit T4 eonversion more effeetively than in the rats of group B. This differenee may weil be due to the difference in age: the rats of group Bare older than those of group A. In eonjunetion with OUr earlier fmding (Wil~rsinga and Touber 1977) of the in vive lowering of plasma levels of T3 by propranolol, the demonstration of the in vitro inhibitory effeet of this drug underlines the importance of ß-adrenoeeptor agonists and antagonists in the metabolism and possibly the ultimate biological effeet of thyroid hormones.
o o
15
30
80
TIME (minut •• )
Fig.4 Conversion of exogenous T4 (1.3 "M) into Tl by isolated rat Iiver ceUs (group B, n = 12; mean protein content ± SD is 18.1 ± 1.1 mg/mI). Conversion ratios are given as mean ±SEM. • - . controls, . - . propranolol 290 "M, . - . proprano101580 "M, 0 - 0 propranolol 1160 "M.
Acknowledgements We are very much indebted to Dr. J. James and C. Schuyt (laboratory of Histology, Jan Swammerdam Instituut, University of Amsterdam) for the preparation of the rat liver cell suspensions. We would also like to thank Dr. H.J.A. SalM (Laboratory of Medical Physics, University of Amsterdam) who performed the statistical analyses. Tbe critical suggestions of Dr. L. Offerhaus and Dr. P.A. van Swieten are gratefuUy acknowledged.
References Abuid, 1., P.R. Lanen: Triiodothyronine and thyroxine in hyperthyroidism. Comparison of the acute changes during therapy with antithyroid agents. J. Clin. Invest. 54: 201-208 (1974) Armitage, P.: Statistical methods in medical research. BlackweU Scic:ntific Publications. Oxford (1971) Bnzverman, L.E., S.H Ingbar, K. Sterling: Conversion of thyroxine (T4) to triiodothyronine (Tl) in athyreotic human subjects. J. Clin. Invest. 49: 855-864 (1970) Burger, A., D. Dinichert, P. Nicod, M. Jenny, Th. Lemarchand-Beraud, M.B. Valloton: Effect of amiodarone on serum trüodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. A drug influencing peripheral metabolism of thyroid hormones, J. CHn. Invest_ 58: 255-259 (1976) Bürgi, H., C. Wimpjheimer, A. Burger, W. Zaun bauer, H. Röf/er, Th. Lemarchand-Beraud: Changes of circulating thyroxine, triiodothyronine and reverse triiodothyronine after radiographie contrast agents. J. Clin. Endocrinol. Metab. 43: 1203-1210 (1976)
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11M.
C.J.F. van Noorden, W.M. Wiersinga and J.L. Touber
Bu", W.A., D.B. Ramsden, R.S. Griffiths, E.G. Black, R. Hoffenberg, H. Meinhold, K. W. Wenzel: Effect of a single dose of dexamethasone on serum concentrations of thyroid hormones. Lancet 1976/11,58-61 Cheung, M.C., W.R. Haunwhite Jr.: Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. Clin. Chem. 22: 299-304 (1976) Chopra, 1.1., D.E. Williams, J. Orgiazzi, D.H. Solomon: Opposite effects of dexamethasone on serum concentrations of 3,3',5'-trüodothyronine (reverse T3) and 3,3',5-trüodothyronine (T3) in man. J.Clin.Endocrinol.Metab. 41: 911-920 (1975) Chopra, 1.1.: An assessment of daily production and significance of thyroidal secretion of 3,3',5'-triiodothyronine (reverse T3) in man. J. Clin. Invest. 58: 32-40 (1976) Chopra, 1.1.: A study of extrathyroidal conversion of thyroxine (T4 ) to 3,3',5-trüodothyronine (T3) in vitro. Endocrinology 101: 453-463 (1977) Fisher, D.A., 1.1. Chopra, J.H. Dussoult: Extrathyroidal conversion of thyroxine to trüodothyronine in sheep. Endocrinology 91: 1141-1144 (1972) Gelfner, D.L., M. Azukizawa, H.M. Hershman: Propylthiouracil blocks extrathyroidal conversion of thyroxine to trüodothyronine and augments thyrotropin secretion in man. J. Clin. Invest. 55: 224-229 (1975) Hayes, A., R. G. Cooper: Studies on the absorption, distribution and excretion of propranolol in rat, dog and monkey. J. Pharmacol. Exp. Ther. 176: 302-311 (1971) Hesch, R.D., G. Brunner, H.D. Söling: Conversion of thyroxine (T4) and trüodothyronine (T3) and the subcellular localisation of the converting enzyme. Clin. Chim. Acta 59: 209-213 (1975) Hüfner, M., M. Knöpfte: Pharmacological influences on T4 to T3 conversion in rat liver. Clin. Chim. Acta 72: 337341 (1976) Hüfner, M., M. Grussendorf, M. Ntokalou: Properties of the thyroxine (T4) monodeiodinating system in rat liver homogenate. Clin. Chim. Acta 78: 251-259 (1977) Itzhaki, R.F., D.M. Gill: A micro-biuret method for estirnating proteins. Anal. Bioehern. 9: 401-410 (1964)
James. J.: The genesis of polyploidy in rat liver parenchymal cells. Cytobiology 15: 410-419 (1977) Nies, A.S., D.G. Shand: Clinical pharmacology of propranolol. Circulation 52: 6-15 (1975) Saberi, M, F.H. Sterling, R.D. Utiger: Reduction in extrathyroidal trüodothyronine produption by propylthiouracil in man. J.C1in. Invest. 55: 218-223 (1975) Schwartz, H.L., M.S. Surks, J.H. Oppenheimer: Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3'-trüodo-L-thyroxine in the rat. J. Clin. Invest. 50: 1124-1130 (1971) Spjr)tvoll, E.: Ordering ordered parameters. Biometrica 64: 327-334 (1977) Visser, Th.l., I. van der Does·Tobe, R. Docter, G. Henneman: Conversion of thyroxine into tri-iodothyronine by rat liver homogenate. Biochem. J. 150: 489-493 (1975) Visser, Th.l., I. van der Does-Tobe, R. Docter, G. Henneman: Subcellular locaHzation of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups. Bioehern. J. 157: 479-482 (1976) Westgren, u., A. Melander, S. Ingemansson, A. Burger, S. Tiblin, E. W8hlin: Secretion of thyroxine, 3,5,3'-triiodothyronine and 3,3',5'-trüodothyronine in euthyroid man. Acta Endocrinol. 84: 281-289 (1977) Westgren, u., A. Melander, E. Wtfhlin, I. Lindgren: Divergent effects of 6-propylthiouracil on 3,5,3'-trüodothyronine (T3) and 3,3',5'-trüodothyronine (RT3) serum levels in man. Acta Endocrinol. 85: 345-350 (1977) Westgren, u., B. Ahren. A. Burger, S. Ingemansson, A. Melander: Effects of dexamathasone, desoxycorticosterone, and ACTH on serum concentrations of thyroxine, 3,5,3'triiodothyronine and 3,3',5'-trüodothyronine. Acta Med. Scand. 202: 89-92 (1977) Wiersinga, W.M., I.L. Touber: The influence of j1-adrenoce~ tor blocking agents on plasma thyroxine and trüodothyronine. J. Clin. Endocrinol. Metab. 45: 293-298 (1977) Wolfe, B.B., T.K. Harden, P.B. Molinoft j1-adrenergic rece~ tors in rat liver: effects of adrenalectomy. Proc. Nat. Acad. Sei. USA 73: 1343-1347 (1976)
Requests for reprints should be addressed to: Dr. W.M. Wiersinga, Division of Endocrinology, Department of Medicine, Wilhelmina Gasthuis, Eerste Helmersstraat 104, Amsterdam (Holland)
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370
Propranolol Inhibits the In Vitro Conversion of Thyroxine into Triiodothyronine by Isolated Rat Liver Parenchymal Cells C.J. F. van Noorden, W.M. Wiersinga and J. L. Touber Division of Endocrinology, Department of Medicine, University Hospital "Wilhelmina Gasthuis", Amster· dam, The Netherlands
vel of T3 in hyperthyroid patients and in hypothyroid patients on L-thyroxine substitution therapy (WierA model for the in vitro study of the conversion of thyroxsinga and Touber 1977). Again, inhibition of the peine into trüodothyronine using isolated rat liver parenchymal ripheral conversion seemed the most likely explanacells is described. Isolated liver cells (mean pro tein content 18 mg/mI) convert tion for tbis finding. Since the liver is a major site approximately 0.8% of 1.3 /.IM exogenously added T4 into of T4 -deiodination the decrease in plasma TJ - and T3 during thirty minutes incubation. Carbimazole (50 /.IM) presumably, in T3 production - could be caused by has no effect on the conversion process, whereas propylthia direct effect of propranolol on the parenchym al ouracil (50 /.IM) inhibits the conversion, The tI-adrenoceptor blocking agent propranolol lowers the conversion ratio when liver cells. However, a decrease in T3 production as added in concentrations of 580 and 1160 /.IM, but has no in- a consequence of a decrease in liver perfusion caushibitory effect when 290 j.!M is added. ed by propranolol (Nies and Shand 1975) seemed equally possible. In order to further elucidate the Key-Words: Jsolated Rat Liver Cells - Propranolol - Con· action of propranolol, we studied the effect of the version of Thyroxine - Triiodothyronine drug on liver cells in vitro. The isolated liver cell Introduction preparation - rather than liver cell homogenate was chosen because the action of propranolol is melt has been demonstrated conclusively that 3,3',5-triiodothyronine (T3 ) is mainly produced extrathyroid- dia ted via blockade of the ß-adrenergic receptors of the liver cell plasma membrane (Wolfe, Harden and ally by deiodination of thyroxine (T4 ). This process, Molino!f 1976). known as the peripheral conversion of T4 , leads to formation of the metabolically active hormone TJ and to the inactive 3,3',5' -T3 (reverse T3 or rTJ ) flJra- Materials and Methods verman, Ingbar and Sterling 1970; Schwartz, Surks Liver cells of two groups of male Wistar rats were used; and Oppenheimer 1971; Fisher, Chopra and Dussault mean weight ± SD are 215 :t 11 g in group A (n = 4) and 1972; Chopra 1976; Westgren, Melander, Ingemans298 :t 18 g in group B (n = 12). The rats were anaesthetized with nembutal and received intravenously 0.2 ml heparin son, Burger, Tibblin and W/lhlin 1977). In man seve(1000 i.u.) dissolved in 0.8 ml 1% NaN02 to prevent blood ral dru~s have been shown to influence the periphecoagulation and vascular contraction. Parenchymalliver cells ral conversion. Propyltbiouracil (Abuid and Larsen were isolated by perfusion of the liver with calcium-free 1974; Saberi, Sterling and Utiger 1975; Geffner, Azu- phosphate buffered Ringer-solution saturated with carbogen kizawa and Hersh17um 1975; Westgren, Melander, Wa- (pH 7.4, 37°C) containing 20 g/I bovine serum albumin lin and Lindgren 1977), dexamethasone (Chopra, Wil- and 0.6. g/I collagenase (Sigma) (James 1977). The isolated cells were suspended in 3 ml of a Krebs-Ringer-bicarbonate liams, Orgiazzi and Solomon 1975; Bu", Ramsden, buffer (pH 7.4, 37°C, containing 2.5 g/I bovine serum alGriffiths, Black, Hoffenberg, Meinhold and Wenzel bumin but no collagenase) at a concentration of 10-20 mg 1976; Westgren, Ahren, Burger, Ingemansson and protein per ml and incubated at 37 oe in slowly rotating tubes. Cell suspensions of each individual rat of group A Melander 1977), amiodarone (Burger, Dinichert, Niwere incubated in duplicate with 1.3 /.IM sodium-L-thyroxcod, Jenny, Lemarchand-Beraud and Valloton 1976), ine (Sigma) in the presence or absence of 50 /.IM carbimaand Na-iopanoate (Bürgi, Wimpjheimer, Burger, zole, 50 /.IM 6-propy1-2-thiouracil or 580 /.IM D,L-propranZaunbauer, Rösler and Lemarchand-Beraud 1976), 0101 (I.c.I.). In the same manner, cells of the rats of group all decrease plasma levels of TJ with a concomitant B were incubated with 1.3 /.IM sodium L-thyroxine with or rise in plasma rTJ . Also, propylthiouracil inhibits without D,L-propranolol at concentrations of 290, 580 and 1160 /.IM respectively. the conversion ofT4 into T3 in rat liver homogenate in vitro (Visser, van der Does-Tobe, Docter and HenT4 and the various drugs were added together to the incubaUon mixture and 500 /.11 sampies were taken from the inneman 1975; Chopra 1977). We have recently demonstrated that the ß-adrenoceptor blocking agent propranolol lowers the plasma leReceived: 21 June 1978 0018-5043/79
0532-0366
cubation mixtures at times 0, 15, 30 and 60 minute!. The sampies were immediately transfened to I ml ice-cold absolute ethanol After centrifugation (2000 g, 15 min, 4°q the clear supernatants were stored at -20°C. As controls,
Accepted: 29 Aug. 1978 S 03.00
©
1979
Georg Thieme Publishers
Downloaded by: NYU. Copyrighted material.
Summuy
Inhibition of in Vitro Conversion of T4 to T3 by Propanolol in Isolated Rat Liver CeUs
367
Table 1 Effect of carbimazole, propylthiouracil and propranolol on the conversion of exogenous T4 (1.3 J'M) into T3 by iso 1ated rat liver cells. Conversion ratios are given as the mean ± SEM (group A, n = 4; mean pro tein content ± SD is 18.9±3.8 mg/mI)
15 30 60
controls
conversion ratio (nm»1 T3 formed / nmol T4 added x 100 %) carbimazole PTU propranolol 50 11M 50 11M 580 11M
0.49 ± 0.10 0.86 ± 0.20 1.06 ± 0.25
-0.05 ± 0.02 -0.03 ± 0.06 -0.03 ± 0.06
0.39 ± 0.13 0.78 t 0.18 1.10 t 0.06
100 ,
liver cells were incubated in the absence of T4, alrld T4 was incubated in the absence of liver cells. The protein concentration of the incubation mixture was determinecl by the biuret method (Itzhaki and Gi111964); the concenlration served as a measure of the number of cells present.
90
!
The T3 and T4 concentrations of the ethanol extracts (diluted if necessary) were measured in duplicate by spedfic radio- Bo immunoassays Wisser et al. 1975; Chopra 1977), using 300 g/l polyethyleneglycol (Cheung and Haunwhite lL 1976) for the separation of the bound and free hormone fuctions. Sampies presenting the standard curve were incubated under identica1 conditions, i.e. with a fmal ethanol conl:entration of 6.7% (v/v). The detection limit of the T3-assar is 0.05 nM, of the T4-assay 1.5 nM. Cross-reactivity of T4 in the T3-assay is 0.1). However, both liver cells; in general approximately 10 nM T3 is 50 ILM propylthiouracil and 580J-LM propranolollowered conversion ratios (p < 0.01). formed after 30 min. incubation.
368
C.J.F. van Noorden, W.M. Wiersinga and J.L. Touber
Table 2 Effect of various concentrations of propranolol on the conversion of exogenous T4 (1.3 "M) into T3 by isolated . rat liver cells. Conversion ratiosaregivenas themean ± SEM (Jroup B, n = 12; mean protein content ± SD is 18.1 ± 1.1 mg/mI) Incubation time in minutes
15 30 60
conversion ratio (nmol T3 formed / nmol T4 added x 100%) propranolol propranolol propranolol 290 pM 580 "M 1160 "M
controls 0.52 0.82 1.47
± ± ±
0.06 0.11 0.20
0.34 0.69 1.46
± ± ±
0.21 ± 0.02 0.55 ± 0.10 1.11 :t 0.13
0.05 0.12 0.16
•
1.5
l
0.26:t 0.05 0.56:t 0.09 0.99:t 0.16
1.5
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0.5
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90 TIME (minut•• )
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eo
30
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Fig. 2 Conversion of exogenous T4 (1.3 "M) into T, by OOlated rat liver cells (pro tein content 7.9 mg/ml)
Fig. 3 Conversion of exogenous T4 (l.3·"M) into T3 by 001ated rat liver cells (group A, n =4; mean protein content ± SD is 18.9 ± 3.8 mg/mi). Conversion ratios are given as mean :t SEM.• - . controls, . - . carbimazole 50"M, 0 - 0 proprylthiouracil SO"M, . - . propranolol S80 "M.
No interactions are found in the experiments of group B (results are given in Table 2 and Figure 4). The interactions - and residual sum of squares were pooled. The analysis was continued by a stepwise multiple comparison procedure for ordered parameters (Spj,tvoll 1977). The level of significance was taken as a=0.05. Addition of 580 and 1160 llM propranolol both resulted in lower conversion ratios, but no difference was found between the 580 and 1160 llM dose. No inhibitory effect on the conversion was found for 290 llM propranolol.
when T4 is incubated without cells. We have applied conversion ratios because the deiodinatins capacity of the liver cells is not saturated at the T4 concentrations used (Hesch, Brunner and Söling 1975;
Discussion Our results indicate that isolated rat liver cells are suitable for the in vitro study of the conversion of T4 into T3 ; T3 generation cannot be detected in the absence of T4 nor is there a change in T3 level
Chopra 1977). Conversion of T4 into T3 by isolated liver cells has been reported previously by Hesch, Brunner and Söling (1975). Using a comparable amount of cells, they found a lower conversion ratio (0.1 %) than in the present study (0.8%). Rat liver homogenate has also been shown to generate T3 from T4 (Visser et a1. 1975; Hüfner and Knöpfte 1976; Visser, van der Does-Tobe, Docter and Hennmuzn 1976; Chopra 1977; Hüfner, Grussendorf and Ntokalou 1977); conversion ratios in this model were somewhat higher dependent on the concentrations of substrate and (cell)protein.
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Inhibition of in Vitro Conversion of T4 to T3 by Propranolol in Isolated Rat Liver Cells
The results of the experiments with the eells of the rats of JrQuP A show quite c1early that whc,reas the addition of earbimazole has no effeet on the eonversion process, propylthiouraeil inhibits Tl-generation. Ifl. This is in aeeordanee with the results of studies using ;; rat Iiver homogenate (Visser et a1. 1975; Chopra 1977)_ ~
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1.5
a:
An effeet of propranolol on the in vitro eonversion of T4 has thus far not been reported. Our data show that tbis ß-adrenoceptor blocking agent also inhibits the eonversion of T4 into Tl by isolated rat liver cells. The effeet of propranolol is not an artefact, sinee the drug did not influenee either the T3 levels of eells ineubated without T4 or the radioimmunoassays of T3 and T4 • We have been unable to establish an unequivocal dose dependent effeet of proprano101. Alth()ugh both 580 and 1160 11M propranolol inhibit the eonversion process while 290 11M propranolol does not, there is no difference between the effeets of 580 alld 1160
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9
:l1.0 111 > Z
I
o
u
0.5
2
Sinee propranolo1 is rapidly metabolised by the 1iver (Nies and Shand 1975) low levels of Jlroprano101 may not be suffieient to initiate or to maintain an inhibitory effect. Tbis possibility might be explored in experiments using ß-adrenoceptor blocking agents wbieh are not metabolised by the liver, such as atenolol and praetolol. The eoncentrations of propranolol used in our ineubation mixtures may have been mueh bigher than in vive portal plasma levels of subjeets given the usual therapeutie dosage. The results of the studies of Hayes and Cooper (1971) indicate that, in the dog, propranolol eoneentrations in liver tis~rue are in the order of 0.5 Ilg/g (about ten times bigher than the eorresponding blood levels) after the administration of 1 mg/kg orally. It is therefore eonceivab1e that in our experiments other than the ß-blocking properties of the drug - such as the membrane stabilising aetivity - may have influeneed the results. Studies with ß-b1ocking agents whieh lack tbis aetivity (e.g. atenolol and metoprolol) are required to further elueidate the meehanism of action in this respect_ In the rats of group A proprano101 seems t() inhibit T4 eonversion more effeetively than in the rats of group B. This differenee may weil be due to the difference in age: the rats of group Bare older than those of group A. In eonjunetion with OUr earlier fmding (Wil~rsinga and Touber 1977) of the in vive lowering of plasma levels of T3 by propranolol, the demonstration of the in vitro inhibitory effeet of this drug underlines the importance of ß-adrenoeeptor agonists and antagonists in the metabolism and possibly the ultimate biological effeet of thyroid hormones.
o o
15
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80
TIME (minut •• )
Fig.4 Conversion of exogenous T4 (1.3 "M) into Tl by isolated rat Iiver ceUs (group B, n = 12; mean protein content ± SD is 18.1 ± 1.1 mg/mI). Conversion ratios are given as mean ±SEM. • - . controls, . - . propranolol 290 "M, . - . proprano101580 "M, 0 - 0 propranolol 1160 "M.
Acknowledgements We are very much indebted to Dr. J. James and C. Schuyt (laboratory of Histology, Jan Swammerdam Instituut, University of Amsterdam) for the preparation of the rat liver cell suspensions. We would also like to thank Dr. H.J.A. SalM (Laboratory of Medical Physics, University of Amsterdam) who performed the statistical analyses. Tbe critical suggestions of Dr. L. Offerhaus and Dr. P.A. van Swieten are gratefuUy acknowledged.
References Abuid, 1., P.R. Lanen: Triiodothyronine and thyroxine in hyperthyroidism. Comparison of the acute changes during therapy with antithyroid agents. J. Clin. Invest. 54: 201-208 (1974) Armitage, P.: Statistical methods in medical research. BlackweU Scic:ntific Publications. Oxford (1971) Bnzverman, L.E., S.H Ingbar, K. Sterling: Conversion of thyroxine (T4) to triiodothyronine (Tl) in athyreotic human subjects. J. Clin. Invest. 49: 855-864 (1970) Burger, A., D. Dinichert, P. Nicod, M. Jenny, Th. Lemarchand-Beraud, M.B. Valloton: Effect of amiodarone on serum trüodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. A drug influencing peripheral metabolism of thyroid hormones, J. CHn. Invest_ 58: 255-259 (1976) Bürgi, H., C. Wimpjheimer, A. Burger, W. Zaun bauer, H. Röf/er, Th. Lemarchand-Beraud: Changes of circulating thyroxine, triiodothyronine and reverse triiodothyronine after radiographie contrast agents. J. Clin. Endocrinol. Metab. 43: 1203-1210 (1976)
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C.J.F. van Noorden, W.M. Wiersinga and J.L. Touber
Bu", W.A., D.B. Ramsden, R.S. Griffiths, E.G. Black, R. Hoffenberg, H. Meinhold, K. W. Wenzel: Effect of a single dose of dexamethasone on serum concentrations of thyroid hormones. Lancet 1976/11,58-61 Cheung, M.C., W.R. Haunwhite Jr.: Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. Clin. Chem. 22: 299-304 (1976) Chopra, 1.1., D.E. Williams, J. Orgiazzi, D.H. Solomon: Opposite effects of dexamethasone on serum concentrations of 3,3',5'-trüodothyronine (reverse T3) and 3,3',5-trüodothyronine (T3) in man. J.Clin.Endocrinol.Metab. 41: 911-920 (1975) Chopra, 1.1.: An assessment of daily production and significance of thyroidal secretion of 3,3',5'-triiodothyronine (reverse T3) in man. J. Clin. Invest. 58: 32-40 (1976) Chopra, 1.1.: A study of extrathyroidal conversion of thyroxine (T4 ) to 3,3',5-trüodothyronine (T3) in vitro. Endocrinology 101: 453-463 (1977) Fisher, D.A., 1.1. Chopra, J.H. Dussoult: Extrathyroidal conversion of thyroxine to trüodothyronine in sheep. Endocrinology 91: 1141-1144 (1972) Gelfner, D.L., M. Azukizawa, H.M. Hershman: Propylthiouracil blocks extrathyroidal conversion of thyroxine to trüodothyronine and augments thyrotropin secretion in man. J. Clin. Invest. 55: 224-229 (1975) Hayes, A., R. G. Cooper: Studies on the absorption, distribution and excretion of propranolol in rat, dog and monkey. J. Pharmacol. Exp. Ther. 176: 302-311 (1971) Hesch, R.D., G. Brunner, H.D. Söling: Conversion of thyroxine (T4) and trüodothyronine (T3) and the subcellular localisation of the converting enzyme. Clin. Chim. Acta 59: 209-213 (1975) Hüfner, M., M. Knöpfte: Pharmacological influences on T4 to T3 conversion in rat liver. Clin. Chim. Acta 72: 337341 (1976) Hüfner, M., M. Grussendorf, M. Ntokalou: Properties of the thyroxine (T4) monodeiodinating system in rat liver homogenate. Clin. Chim. Acta 78: 251-259 (1977) Itzhaki, R.F., D.M. Gill: A micro-biuret method for estirnating proteins. Anal. Bioehern. 9: 401-410 (1964)
James. J.: The genesis of polyploidy in rat liver parenchymal cells. Cytobiology 15: 410-419 (1977) Nies, A.S., D.G. Shand: Clinical pharmacology of propranolol. Circulation 52: 6-15 (1975) Saberi, M, F.H. Sterling, R.D. Utiger: Reduction in extrathyroidal trüodothyronine produption by propylthiouracil in man. J.C1in. Invest. 55: 218-223 (1975) Schwartz, H.L., M.S. Surks, J.H. Oppenheimer: Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3'-trüodo-L-thyroxine in the rat. J. Clin. Invest. 50: 1124-1130 (1971) Spjr)tvoll, E.: Ordering ordered parameters. Biometrica 64: 327-334 (1977) Visser, Th.l., I. van der Does·Tobe, R. Docter, G. Henneman: Conversion of thyroxine into tri-iodothyronine by rat liver homogenate. Biochem. J. 150: 489-493 (1975) Visser, Th.l., I. van der Does-Tobe, R. Docter, G. Henneman: Subcellular locaHzation of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups. Bioehern. J. 157: 479-482 (1976) Westgren, u., A. Melander, S. Ingemansson, A. Burger, S. Tiblin, E. W8hlin: Secretion of thyroxine, 3,5,3'-triiodothyronine and 3,3',5'-trüodothyronine in euthyroid man. Acta Endocrinol. 84: 281-289 (1977) Westgren, u., A. Melander, E. Wtfhlin, I. Lindgren: Divergent effects of 6-propylthiouracil on 3,5,3'-trüodothyronine (T3) and 3,3',5'-trüodothyronine (RT3) serum levels in man. Acta Endocrinol. 85: 345-350 (1977) Westgren, u., B. Ahren. A. Burger, S. Ingemansson, A. Melander: Effects of dexamathasone, desoxycorticosterone, and ACTH on serum concentrations of thyroxine, 3,5,3'triiodothyronine and 3,3',5'-trüodothyronine. Acta Med. Scand. 202: 89-92 (1977) Wiersinga, W.M., I.L. Touber: The influence of j1-adrenoce~ tor blocking agents on plasma thyroxine and trüodothyronine. J. Clin. Endocrinol. Metab. 45: 293-298 (1977) Wolfe, B.B., T.K. Harden, P.B. Molinoft j1-adrenergic rece~ tors in rat liver: effects of adrenalectomy. Proc. Nat. Acad. Sei. USA 73: 1343-1347 (1976)
Requests for reprints should be addressed to: Dr. W.M. Wiersinga, Division of Endocrinology, Department of Medicine, Wilhelmina Gasthuis, Eerste Helmersstraat 104, Amsterdam (Holland)
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